Uptake of acetaldehyde-modified (ethylated) low-density lipoproteins by mouse peritoneal macrophages

Alcohol 26 (2002) 163–166

Uptake of acetaldehyde-modified (ethylated) low-density lipoproteins by mouse peritoneal macrophages Hanna Wehra,*, Ewa Mirkiewiczb, Maria Rodoa, Malgorzata Bednarska-Makaruka a

Department of Genetics, Institute of Psychiatry and Neurology, Sobieskiego 1/9, 02-957 Warsaw, Poland b Department of Biochemistry, 2nd Medical Faculty, Academy of Medicine, Warsaw, Poland Received 3 April 2001; received in revised form 19 November 2001; accepted 12 December 2001

Abstract The uptake of acetaldehyde-modified (ethylated) low-density lipoproteins (LDLs) by murine peritoneal macrophages is described and compared with the uptake of acetylated LDLs. The fluorescent marker DiI was used. No competition between ethylated and acetylated LDLs was observed. Ethylated LDL uptake was not inhibited by polyinosinic acid or fucoidin. Our conclusion is that uptake of ethylated and acetylated LDLs can be done by two different receptors. © 2002 Elsevier Science Inc. All rights reserved. Keywords: Ethylated LDL uptake; Scavenger receptor

1. Introduction The uptake of nonmodified low-density lipoproteins (LDLs) is mediated by an LDL receptor that is down-regulated by the increase of cellular cholesterol content. Low-density lipoproteins become easily modified by various agents, and the uptake of such particles is mediated by scavenger receptors. Scavenger receptors are not regulated, and cells overloaded with cholesterol can accumulate and lead to organ pathology. An example is oxidative LDL modification, which is believed to initiate the atherogenic process in arteries (Steinberg et al., 1989). Out of various scavenger receptors present in macrophages the best known are SR-A I and II. They bind mostly oxidized and acetylated LDLs. In 1986, Horiuchi et al. described a receptor for aldehyde-modified proteins (Horiuchi et al., 1986). Possible natural ligands for this receptor were regarded to be proteins modified by advanced glycation end products (Takata et al., 1988). Many other scavenger receptors (defined as class B or C scavenger receptors) binding modified proteins were described (Yamada et al., 1998). In the course of metabolism small amounts of acetaldehyde are constantly produced (Ma et al., 1989). Its production is very much enhanced after alcohol consumption. The presence of acetaldehyde-modified (ethylated) LDLs in serum samples obtained from alcoholics after a period of intense drinking was described before (Wehr et al., 1993). Eth* Corresponding author. Tel.: 48 22 8427650; fax: 48 22 8589169. E-mail address: [email protected] (H. Wehr). Editor: S. Borg

ylated LDLs were also found in the liver (Lin et al., 1995; Niemela et al., 1994). The mechanism of ethylated LDL uptake and removal is not known. In the present work the possibility that they are natural ligands for macrophage scavenger receptors is studied. Low-density lipoproteins were marked with fluorescent marker DiI, and their uptake was studied by using macrophages isolated from mouse peritoneal cavity. 2. Methods 2.1. Material Male albino Swiss mice ca. 30 g weight were maintained on a normal chow diet (n  180). The experimental protocols were in accordance with the European Communities Council Directive of 24 November 1986 (86/609 EEC) based on the Helsinki Declaration. The experimental procedures were approved by the Ethical Committee of Institute of Psychiatry and Neurology. Human blood for LDL isolation was collected from healthy human subjects (n  10) in EDTA tubes (1 mg/ml blood). Written informed consent was obtained from the subjects. Low-density lipoproteins preparation, marking with DiI, and modification, were performed for each experiment. 2.2. LDL isolation Low-density lipoproteins were isolated by ultracentrifugation according to Havel et al. (1955) in the presence of EDTA, dialyzed against phosphate-buffered saline (PBS) containing EDTA, and stored for 1 day at 4C.

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2.3. LDL marking with fluorescent marker

3. Results

The fluorescent marker 1,1-diooctadecyl 3,3,3,3-tetramethylindocarbocyanine perchlorate (DiI) (Molecular Probes Inc., Eugene, OR, USA) was used. Marking with DiI was done according to the Reynolds and St. Clair procedure (Reynolds & St. Clair, 1985). After incubation and ultracentrifugation, the upper layer containing DiI-LDL was separated and dialyzed as before, filtered through Millipore filters, stored at 4C, and modified the next day.

Comparison of DiI-LDL uptake of ethylated and acetylated LDLs by murine peritoneal macrophages is shown in Fig. 1. The higher uptake was observed in the case of acetylated LDL. Acetylated LDLs in a concentration of 20–200 g/ml medium did not inhibit ethylated DiI-LDL uptake. Ethylated LDLs in a concentration of 20–400 g/ml medium did not inhibit acetylated DiI-LDL uptake. In Fig. 2 the effect of inhibitors—polyinosinic acid and fucoidin—on the uptake of modified LDLs is presented. Four hundred micrograms per dish of the inhibitors was added (final concentration, 267 g/ml medium). It can be seen that acetylated LDL uptake was inhibited by both inhibitors—inhibition by polyinosinic acid and fucoidin in 20 g/ml medium concentration of acetylated LDL protein was 74.5% and 76.8%, respectively. Concerning ethylated LDLs, neither agent inhibited but rather substantially enhanced their uptake. In 20 g/ml medium concentration of ethylated LDL protein polyinosinic acid enhanced their uptake 3.4 times and fucoidin enhanced their uptake 4.1 times.

2.4. LDL modification The DiI-LDLs were ethylated by using acetaldehyde according to Steinbrecher et al. (1984). Acetaldehyde was added in the cold to 1 mg of LDL protein to a final concentration of 320 mM. The hermetically sealed vial was incubated for 45 min at 37C with constant mixing. The DiI-LDLs were acetylated according to Basu et al. (1976). To 1 mg of LDL protein in an ice bath saturated natrium acetate and subsequently 20-l portions of acetic anhydride were added in 20min intervals with constant mixing. Modified LDLs were dialyzed 24 h against several portions of PBS, filtered through Millipore filters, stored at 4C, and used the next day for the experiments. Electrophoretic mobility of modified DiI-LDL was enhanced compared with the nonmodified ones. 2.5. Macrophages isolation

4. Discussion The pathogenic role of ethylated LDLs has not been established so far. It has been shown that highly ethylated LDLs were not bound by the regular LDL receptor (Kerv-

Peritoneal macrophages were harvested in PBS. The perfusate was centrifuged at 400g at 4C for 10 min in sterilized siliconated glass tubes. 2.6. Cell culture studies The cells were washed with MEM supplemented with gentamicin (80 mg/l), resuspended in the same medium containing 10% fetal calf serum (FCS), and dispersed on NUNCLON dishes. After 2 h, the adhering cells were rinsed three times with the medium that did not contain FCS, cultured 18 h in the medium containing FCS, rinsed as before, and used for the experiments. Modified LDLs were added for 2 h in a 1.5-ml total volume of the medium without FCS. The uptake of LDLs was determined after washing the cells twice with the medium containing 0.4% calf albumin, three times with a medium without albumin, and dissolving them in the lysis reagent according to Teupser et al. (1996). In the solution fluorescence was measured at 578 nm by using excitation at 520 nm and a Perkin-Elmer LS-5B spectrofluorimeter. In the same solution protein content was determined. Fluorescence of the LDL preparations was measured in 200-fold diluted solutions, and their specific fluorescence was calculated. Protein was determined by the method of Lowry et al. (1951) by using a Kashyap et al. (1980) modification. The results were expressed as nanograms of LDL protein taken by the cells per milligram of cell protein.

Fig. 1. Comparison of the uptake of acetylated and ethylated low-density lipoproteins (LDLs) by murine peritoneal macrophages. Low-density lipoproteins were isolated by ultracentrifugation, marked with DiI, modified in vitro—acetylated or ethylated—and their 2-h uptake by mouse peritoneal macrophages was measured. Each dish contained 10–50 g/ml medium of modified LDLs in 1.5-ml total volume of MEM without fetal calf serum (FCS). Cells were washed and dissolved in SDS/NaOH according to Teupser et al. (1996), and their fluorescence and protein content were measured.

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Fig. 2. Comparison of the effect of inhibitors on the uptake of variously modified low-density lipoproteins (LDLs). Inhibitors: polyinosinic acid (5) potassium salt (Sigma [PI]) and fucoidin Fucus Vesiculosus (Sigma [FVC]). Samples with inhibitors contained polyinosinic acid or fucoidin in final concentration 267 g/ml medium. (A) Effect of polyinosinic acid on acetylated DiI-LDL uptake. (B) Effect of fucoidin on acetylated DiI-LDL uptake. (C) Effect of polyinosinic acid on ethylated DiI-LDL uptake. (D) Effect of fucoidin on ethylated DiI-LDL uptake. The LDLs were isolated by ultracentrifugation, marked with DiI, modified in vitro— acetylated or ethylated—and their 2-h uptake by mouse peritoneal macrophages was measured. Each dish contained 10–50 g/ml medium of modified LDL in 1.5 ml total volume of MEM without fetal calf serum (FCS). Cells were washed and dissolved in SDS/NaOH according to Teupser et al. (1996), and their fluorescence and protein content were measured.

inen et al., 1991). Their binding by scavenger receptors of reticuloendothelial cells has not been studied before. This work demonstrates that ethylated LDLs had an uptake by mouse peritoneal macrophages. No competition between ethylated and acetylated LDLs was observed. Ethylated LDL uptake was not inhibited by polyinosinic acid or fucoidin, known as inhibitors of class A scavenger receptors. On the contrary, both agents enhanced ethylated LDL uptake; the mechanism of this enhancement is not known. The results support the suggestion that the receptor we found played an active role in ethylated LDL uptake but was not of the class A type (Steinbrecher, 1999; Yamada et al., 1998). This receptor differed also in respect to its reaction with inhibitors from the receptor described by Horiuchi et al. (1986). It was observed that moderate alcohol drinking played an advantageous role in atherosclerosis. On the contrary, in heavy drinking, atherosclerosis is enhanced (Criqui, 1999; Klatsky, 1994), and it is not known what the main factor is in its development. It should be emphasized that modification of lipoproteins with acetaldehyde primarily takes place in the liver (Wehr et al., 1993). The LDL ethylation could, therefore, exert its harmful effect in the development of

postalcoholic liver disease more than that of atherosclerosis. The role of LDL modification by acetaldehyde and uptake of LDLs by the arterial wall needs further study.

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